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CYP1A2 activity is an important determinant of clozapine dosage in schizophrenic patients.

Clozapine is an effective atypical antipsychotic drug applied in the treatment of resistant schizophrenia. The drug is mainly metabolized by cytochrome P-450 (CYP) enzymes especially the isozyme CYP1A2. Remarkably, the effective dosage varies widely among patients, making it necessary to individualize drug therapy with clozapine. The explanation for dosage variation may be differences in drug metabolism, and more specifically of CYP1A2 activity. This study is aimed at determining to what extent variability in clozapine dose can be explained by pharmacokinetic (PK) factors and more specifically by CYP1A2 activity in effectively treated psychiatric patients. In 22 evaluable patients with a schizophrenic disorder chronically using clozapine, the CYP1A2 activity and the clozapine clearance were estimated. For calculation of the pharmacokinetic parameters of clozapine, population PK software based upon Bayesian analysis was used. Caffeine clearance was estimated with the paraxanthine/caffeine ratio and served as estimate of CYP1A2 activity.A significant linear relationship was found between the clozapine dose and clozapine clearance (R: 0.71; P<0.05), whereas no relationship was found between clozapine dosage and clozapine serum trough concentration. Moreover, individual caffeine and clozapine clearances were found to be significantly related (R: 0.62; P<0.05) as were clozapine dose per kg body weight and P/C mol ratio (R: 0.44; P<0.05). We conclude that CYP1A2 activity is an important determinant of the variability of effective clozapine doses in psychiatric patients.

Adult↗

The effect of interleukin-1 on cytokine gene expression by human corneal epithelial cells.

The purpose of this study was to characterize the pattern of cytokine gene expression by human corneal epithelial cells (HCEC) in response to interleukin-1 (IL-1). Primary cultured HCEC (P-HCEC) or SV40 transformed HCEC (SV40-HCEC) were treated for 6 hr with serum-free growth-media alone or with recombinant human IL-1beta or IL-1alpha (10 ng ml(-1)). 33P labeled cDNA was generated from total RNA, then hybridized to a human cytokine expression array. An autoradiograph was generated for each experimental condition and results analysed semi-quantitatively. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect mRNA for IL-8, growth related oncogene-beta (GRO-beta), intercellular adhesion molecule (ICAM)-1 and Ephrin A5. P-HCEC and SV40-HCEC demonstrated comparable cytokine profiles. For P-HCEC (n=2) the expression of 35 genes was upregulated or only detectable following IL-1beta treatment whereas the expression of nine genes was downregulated or undetectable after IL-1beta treatment. In SV40-HCEC (n=3), the expression of 48 genes was upregulated or only detectable following IL-1beta treatment and the expression of 10 genes was downregulated or undetectable after IL-1beta treatment. Some genes that demonstrated increased expression included cadherin-5, ICAM-1, GRO-alpha, GRO-beta, GRO-gamma, Activin A (bA subunit), tumor necrosis factor-alpha, IL-6, and IL-8. Genes that showed decreased expression included the chemokine receptor-CXCR-4, ciliary neurotrophic factor (CNTF), c-kit ligand, Ephrin A5, G-protein coupled receptor RDC-1 and FGF family FGFR2. Bayesian analysis of the SV40-HCEC data (n=3) revealed the expression of 15 genes that were significantly (p<0.05) differentially regulated. Within these 15 genes, the expression of chemokines (GRO-alpha, GRO-beta, IL-8), fibroblast growth factor 13 and the cytokine IL-6 were the most upregulated, while ephrin A5 and chemokine receptor-4 were the most downregulated. IL-1alpha treatment (n=1 P-HCEC; n=1 SV40-HCEC) produced results very similar to IL-1beta treatment. RT-PCR revealed differential regulation of IL-8, GRO-beta, ICAM-1 and ephrin A5 in accordance with gene array data. In conclusion, the data demonstrate that IL-1 treatment of HCEC differentially regulates the expression of other cytokine and related genes, thus adding to the body of evidence that IL-1 is a major mediator of ocular surface inflammatory reactions. Since the expression of a large number of genes can be studied simultaneously, gene array studies such as these offers the advantage of understanding global changes in response to a specific stimulus. Thus our study provides insight in to the ocular surface response in conditions of inflammation and corneal wound healing where the levels of IL-1 are known to be increased.

Cells, Cultured↗

Molecular and karyological aspects of Batoidea (Chondrichthyes, Elasmobranchi) phylogeny.

Although considerable progress has been made in elucidating the relationships within the Chondrichthyes, there is no agreement as it concerns the systematics of Batoidea, the most derived superorder among cartilaginous fishes, and many different interpretations exist. Our investigation provides the first assessment of relationships among the described batoid species using sequences from both mtDNA and nuclear genes as well as karyological morphology. Our work consists primarily in reconstructing the phylogenetic relationships of Batoidea by examining the mtDNA (16S) and nuclear gene (18S) sequences from 11 batoid species. The three analytical methods (NJ, MP and Bayesian analysis) grouped Rajiformes, Myliobatiformes and Rhinobatiformes. In these trees the two torpedoes diverge from the other batoid fishes. We also compare the molecular data with the available karyological evidence, which consist of the diploid number and the karyotype morphology of eight species belonging to the four orders examined. The results show that the karyological structure in the different species is generally consistent with the various phylogenetical trees, and that Torpediniformes confirm their unique genome organization.

Animals↗

A Bayesian approach for estimating values for prevalence and diagnostic test characteristics of porcine cysticercosis.

Several diagnostic techniques are used to estimate the prevalence of the zoonotic tapeworm Taenia solium in pigs, but none of these tests are perfect, making interpretation of results difficult. A Bayesian approach was used to estimate values for the prevalence and diagnostic test characteristic of porcine cysticercosis by combining results of four imperfect tests. Village pigs (N = 868) slaughtered in Lusaka (Zambia), were bled, and tongue and routine meat inspected; and serum antibody and parasite antigen concentrations were determined by ELISA. A model, based on a multinomial distribution and including all possible interactions between the individual tests required 31 parameters to be estimated, but actually allowed only 15 parameters (i.e. had 15 degrees of freedom) to be estimated. Therefore, prior expert opinion on specificity and (in)-dependence of the tests was entered in the model, resulting in a reduction of the number of parameters to be estimated. The estimated prevalence of porcine cysticercosis was 0.642 (95% confidence interval 0.54-0.91). The performances of the tests were (sensitivity (se)-specificity (sp)): tongue inspection (se 0.210-sp 1.000), meat inspection (se 0.221-sp 1.000), Ab-ELISA (se 0.358-sp 0.917), Ag-ELISA (se 0.867-sp 0.947). To validate the estimates obtained from the model we performed a second study: 65 randomly purchased Zambian village pigs were bled for serum antibody and antigen determination, their tongue and meat inspected; and in addition, the carcasses were dissected for total cysticercus counts (gold standard). Cysticerci were found in 31 pigs (prevalence 0.477, 95% confidence interval 0.35-0.60), overlapping with the estimated prevalence in the first study. Sensitivity and specificity values obtained for the aforementioned tests in this study were in agreement with those estimated. A Bayesian analysis framework offers the possibility to combine prior opinion with experimental data to more accurately estimate the real prevalence of porcine cysticercosis in the absence of a gold standard.

Animals↗

Prognostic index score and clinical prediction model of local regional recurrence after mastectomy in breast cancer patients.

PURPOSE: To develop clinical prediction models for local regional recurrence (LRR) of breast carcinoma after mastectomy that will be superior to the conventional measures of tumor size and nodal status. METHODS AND MATERIALS: Clinical information from 1,010 invasive breast cancer patients who had primary modified radical mastectomy formed the database of the training and testing of clinical prognostic and prediction models of LRR. Cox proportional hazards analysis and Bayesian tree analysis were the core methodologies from which these models were built. To generate a prognostic index model, 15 clinical variables were examined for their impact on LRR. Patients were stratified by lymph node involvement (<4 vs. >or =4) and local regional status (recurrent vs. control) and then, within strata, randomly split into training and test data sets of equal size. To establish prediction tree models, 255 patients were selected by the criteria of having had LRR (53 patients) or no evidence of LRR without postmastectomy radiotherapy (PMRT) (202 patients). RESULTS: With these models, patients can be divided into low-, intermediate-, and high-risk groups on the basis of axillary nodal status, estrogen receptor status, lymphovascular invasion, and age at diagnosis. In the low-risk group, there is no influence of PMRT on either LRR or survival. For intermediate-risk patients, PMRT improves LR control but not metastases-free or overall survival. For the high-risk patients, however, PMRT improves both LR control and metastasis-free and overall survival. CONCLUSION: The prognostic score and predictive index are useful methods to estimate the risk of LRR in breast cancer patients after mastectomy and for estimating the potential benefits of PMRT. These models provide additional information criteria for selection of patients for PMRT, compared with the traditional selection criteria of nodal status and tumor size.

Adult↗

A control study to evaluate a computer-based microarray experiment design recommendation system for gene-regulation pathways discovery.

The main topic of this paper is evaluating a system that uses the expected value of experimentation for discovering causal pathways in gene expression data. By experimentation we mean both interventions (e.g., a gene knock-out experiment) and observations (e.g., passively observing the expression level of a "wild-type" gene). We introduce a system called GEEVE (causal discovery in Gene Expression data using Expected Value of Experimentation), which implements expected value of experimentation in discovering causal pathways using gene expression data. GEEVE provides the following assistance, which is intended to help biologists in their quest to discover gene-regulation pathways: Recommending which experiments to perform (with a focus on "knock-out" experiments) using an expected value of experimentation (EVE) method. Recommending the number of measurements (observational and experimental) to include in the experimental design, again using an EVE method. Providing a Bayesian analysis that combines prior knowledge with the results of recent microarray experimental results to derive posterior probabilities of gene regulation relationships. In recommending which experiments to perform (and how many times to repeat them) the EVE approach considers the biologist's preferences for which genes to focus the discovery process. Also, since exact EVE calculations are exponential in time, GEEVE incorporates approximation methods. GEEVE is able to combine data from knock-out experiments with data from wild-type experiments to suggest additional experiments to perform and then to analyze the results of those microarray experimental results. It models the possibility that unmeasured (latent) variables may be responsible for some of the statistical associations among the expression levels of the genes under study. To evaluate the GEEVE system, we used a gene expression simulator to generate data from specified models of gene regulation. Using the simulator, we evaluated the GEEVE system using a randomized control study that involved 10 biologists, some of whom used GEEVE and some of whom did not. The results show that biologists who used GEEVE reached correct causal assessments about gene regulation more often than did those biologists who did not use GEEVE. The GEEVE users also reached their assessments in a more cost-effective manner.

Animals↗

Spliced leader RNA gene promoter sequence heterogeneity in CL-Brener Trypanosoma cruzi reference strain.

Trypanosoma cruzi is divided into two phylogenetic lineages, T. cruzi I and T. cruzi II, which contain different spliced leader (SL) RNA gene promoter sequences: Class I SL gene promoter sequences are found in T. cruzi II, and Class II sequences in T. cruzi I. We analysed different SL RNA promoter sequences from CL-Brener reference strain, belonging to T. cruzi II lineage, and detected sequences that differed within the -80/+1 highly conserved region. Indeed, many of these divergent SL promoters present features of T. cruzi I promoters. Some of these sequences were grouped into the T. cruzi I sequences clade by Bayesian analysis. The results presented herein show that sequence heterogeneity in SL RNA gene promoter not only exists between T. cruzi strains but also within CL-Brener strain. These CL-Brener "T. cruzi I-like" sequences could be considered a molecular trace of a hybrid origin of the SL RNA gene and a new evidence for the presence of sequences of T. cruzi I origin into a T. cruzi II strain. The possible origins of these sequences are discussed.

Animals↗

Phylogeny and phenotypic variation in the lichen family Graphidaceae (Ostropomycetidae, Ascomycota).

A phylogenetic study of the lichen family Graphidaceae is presented. Most genera of the family, as well as selected representatives of the closely related Thelotremataceae, are included. The results of the Bayesian analysis of combined mt SSU and nuLSU rDNA sequence data were compared with recently introduced concepts of genera. Species of Fissurina and Dyplolabia form a monophyletic group in an unresolved sister-group relationship to other members of Graphidaceae and Thelotremataceae. The family Graphidaceae as currently circumscribed is paraphyletic and we suggest that the name Graphidaceae is used in a broader sense to include members of Thelotremataceae. The concepts of Glyphis, Phaeographis and Platygramme are confirmed by molecular data. Surprisingly, Graphis species are found in two distinct clades, which can only partly be explained by morphology. Hemithecium as recently circumscribed is polyphyletic: H. implicatum, with hyaline spores groups within the Graphis scripta clade, whereas a second Hemithecium species, representing subgenus Leucogramma with brown spores, forms a well-supported clade with other brown-spored species such as 'Sarcographina'lyphiza, Leiorreuma hypomelaenum and Sarcographa ramificans. The evolutionary pattern of morphological characters of the ascomata such as exciple carbonization, paraphyses types, and ascospore characters (colour, septation, and Lugols reaction) are critically discussed.

Ascomycota↗

Partial correlation for functional brain interactivity investigation in functional MRI.

Examination of functional interactions through effective connectivity requires the determination of three distinct levels of information: (1) the regions involved in the process and forming the spatial support of the network, (2) the presence or absence of interactions between each pair of regions, and (3) the directionality of the existing interactions. While many methods exist to select regions (Step 1), very little is available to complete Step 2. The two main methods developed so far, structural equation modeling (SEM) and dynamical causal modeling (DCM), usually require precise prior information to be used, while such information is sometimes lacking. Assuming that Step 1 was successfully completed, we here propose a data-driven method to deal with Step 2 and extract functional interactions from fMRI datasets through partial correlations. Partial correlation is more closely related to effective connectivity than marginal correlation and provides a convenient graphical representation for functional interactions. As an instance of brain interactivity investigation, we consider how simple hand movements are processed by the bihemispheric cortical motor network. In the proposed framework, Bayesian analysis makes it possible to estimate and test the partial statistical dependencies between regions without any prior model on the underlying functional interactions. We demonstrate the interest of this approach on real data.

Cerebral Cortex↗

Body surface mapping versus the standard 12 lead ECG in the detection of myocardial infarction amongst emergency department patients: a Bayesian approach.

OBJECTIVE: To determine if body surface mapping (BSM) is better than the standard 12 lead ECG in the diagnosis of acute myocardial infarction amongst emergency department patients. SETTING: A University affiliated inner-city emergency department. PARTICIPANTS: People presenting to an emergency department with symptoms compatible with myocardial ischaemia/infarction. MAIN OUTCOME MEASURES: Myocardial infarction as defined by either standard 12 lead ECG changes with associated cardiac marker rise, Troponin T >0.1 microg/ml at > 12 h or autopsy/surgical findings of fresh macroscopic infarction. RESULTS: BSM had an overall sensitivity of 47.1% versus 40% for the 12 lead ECG (P < 0.001). Specificity for the BSM was 85.6% versus 93.7% for the 12 lead ECG (P < 0.001). These findings were consistent for low/moderate and high risk subgroups. Bayesian analysis demonstrates that indiscriminate use of BSM would result in a clinically important overdiagnosis of myocardial infarction amongst emergency department patients. CONCLUSIONS: BSM has a higher sensitivity, but a lower specificity for the diagnosis of myocardial infarction.

Adult↗

Can cognitive processes be inferred from neuroimaging data?

There is much interest currently in using functional neuroimaging techniques to understand better the nature of cognition. One particular practice that has become common is 'reverse inference', by which the engagement of a particular cognitive process is inferred from the activation of a particular brain region. Such inferences are not deductively valid, but can still provide some information. Using a Bayesian analysis of the BrainMap neuroimaging database, I characterize the amount of additional evidence in favor of the engagement of a cognitive process that can be offered by a reverse inference. Its usefulness is particularly limited by the selectivity of activation in the region of interest. I argue that cognitive neuroscientists should be circumspect in the use of reverse inference, particularly when selectivity of the region in question cannot be established or is known to be weak.

Brain↗

Preliminary phylogeny of Valerianaceae (Dipsacales) inferred from nuclear and chloroplast DNA sequence data.

Valerianaceae is a relatively small (ca. 350 species), but morphologically diverse angiosperm clade. Sequence data from the entire ndhF gene, the trnL-F intergenic spacer region, the trnL intron, the matK region, the rbcL-atpB intergenic spacer region and internal transcribed spacer (ITS) region of nuclear ribosomal DNA were collected for 21 taxa within Dipsacaceae and Valerianaceae (1 and 20, respectively). These data were included in several phylogenetic analyses with previously published sequences from Dipsacales. Results from these analyses (maximum parsimony, maximum likelihood, and Bayesian analysis) are in strong agreement with many of the conclusions from previous studies, most importantly: (1) Valerianaceae is sister to Dipsacaceae; (2) Triplostegia is more closely related to species of Dipsacaceae than to Valerianaceae; and (3) Valeriana appears not to be monophyletic, with Valeriana celtica falling outside the remainder of the species of Valeriana sampled here (with very strong support). With the exception of V. celtica, these data support two major clades within Valeriana; one that is exclusively New World and another that is distributed in both the Old and New World. Although the species of Valerianaceae and its sister group Dipsacaceae plus Triplostegia, are widely distributed in the Northern Hemisphere, and the data imply that Valerianaceae diversified initially in Asia (the Himalayan Patrinia and Nardostachys falling at the base of the clade), the center of modern species diversity for the group is in the Andes of South America with as many as 175 species restricted to that region. Although the exclusively South American taxa form a clade in the chloroplast and combined ITS and chloroplast analyses, support values tend to be low. Future studies will need to include additional data, in the form of both characters and taxa, before any strong conclusions about the character evolution, diversification, and biogeography of the South American valerians can be made.

Bayes Theorem↗

Phylogenetic relationships of Iberian Aphodiini (Coleoptera: Scarabaeidae) based on morphological and molecular data.

A phylogeny of Iberian Aphodiini dung beetles was reconstructed based on morphological and molecular data. The data set included a total of 84 variable characters from wing venation, mouthparts, genitalia, and external morphology, as well as mitochondrial partial cytochrome c oxidase I (COI), complete tRNA-Leu (UUR), and partial cytochrome c oxidase II (COII) gene nucleotide sequences (1210 positions). Phylogenetic trees based on molecular data were relatively more resolved than those based on morphological characters. The Bayesian analysis of combined molecular and morphological data provided resolution not achieved by each data set separately. Ammoecius and Aphodius are the first lineages that branch off from the tree, followed by Acrossus, Nimbus, and Heptaulacus. The remaining studied taxa are recovered in a more derived clade that lacks internal resolution. Reconstructed trees based on molecular data showed relatively short internal nodes that were weakly supported. Such pattern may reflect a rapid radiation at the origin of the tribe Aphodiini, but also saturation of mutational changes. Several tests were conducted to discern between both competing hypotheses, as well as to assess the effect of incomplete taxon sampling.

Animals↗

Relict snakes of North America and their relationships within Caenophidia, using likelihood-based Bayesian methods on mitochondrial sequences.

This paper focuses on the phylogenetic relationships of eight North American caenophidian snake species (Carphophis amoena, Contia tenuis, Diadophis punctatus, Farancia abacura, Farancia erytrogramma, Heterodon nasicus, Heterodon platyrhinos, and Heterodon simus) whose phylogenetic relationships remain controversial. Past studies have referred to these "relict" North American snakes either as colubrid, or as Neotropical dipsadids and/or xenodontids. Based on mitochondrial DNA ribosomal gene sequences and a likelihood-based Bayesian analysis, our study suggests that these North American snakes are not monophyletic and are nested within a group (Dipsadoidea) that contains the Dipsadidae, Xenodontidae, and Natricidae. In addition, we use the relationships proposed here to highlight putative examples of parallel evolution of hemipenial morphology among snake clades.

Animals↗

Snakes across the Strait: trans-Torresian phylogeographic relationships in three genera of Australasian snakes (Serpentes: Elapidae: Acanthophis, Oxyuranus, and Pseudechis).

We analyze the phylogeny of three genera of Australasian elapid snakes (Acanthophis-death adders; Oxyuranus-taipans; Pseudechis-blacksnakes), using parsimony, maximum likelihood, and Bayesian analysis of sequences of the mitochondrial cytochrome b and ND4 genes. In Acanthophis and Pseudechis, we find evidence of multiple trans-Torresian sister-group relationships. Analyses of the timing of cladogenic events suggest crossings of the Torres Strait on several occasions between the late Miocene and the Pleistocene. These results support a hypothesis of repeated land connections between Australia and New Guinea in the late Cenozoic. Additionally, our results reveal undocumented genetic diversity in Acanthophis and Pseudechis, supporting the existence of more species than previously believed, and provide a phylogenetic framework for a reinterpretation of the systematics of these genera. In contrast, our Oxyuranus scutellatus samples from Queensland and two localities in New Guinea share a single haplotype, suggesting very recent (late Pleistocene) genetic exchange between New Guinean and Australian populations.

Animals↗

A mitochondrial phylogeny of the rainforest skink genus Saproscincus, Wells and Wellington (1984).

The phylogenetic relationships and historical biogeography of 10 currently described rainforest skinks in the genus Saproscincus were investigated using mitochondrial protein-coding ND4 and ribosomal RNA 16S genes. A robust phylogeny is inferred using both maximum likelihood and Bayesian analysis, with all inter-specific nodes strongly supported when datasets are combined. The phylogeny supports the recognition of two major lineages (northern and southern), each of which comprises two divergent clades. Both northern and southern lineages have comparably divergent representatives in mid-east Queensland (MEQ), providing further molecular evidence for the importance of two major biogeographic breaks, the St. Lawrence gap and Burdekin gap separating MEQ from southern and northern counterparts respectively. Vicariance associated with the fragmentation and contraction of temperate rainforest during the mid-late Miocene epoch underpins the deep divergence between morphologically conservative lineages in at least three instances. In contrast, one species, Saproscincus oriarus, shows very low sequence divergence but distinct morphological and ecological differentiation from its allopatric sister clade within Saproscincus mustelinus. These results suggest that while vicariance has played a prominent role in diversification and historical biogeography of Saproscincus, divergent selection may also be important.

Animals↗

Molecular systematics of the butterfly genus Ithomia (Lepidoptera: Ithomiinae): a composite phylogenetic hypothesis based on seven genes.

Butterflies in the nymphalid subfamily Ithomiinae are brightly colored and involved in mimicry. Here we present a phylogenetic hypothesis for 23 of the 24 species in the genus Ithomia, based on seven different gene regions, representing 5 linkage groups and 4469 bp. We sequenced varying length regions of the following genes: (1) elongation factor 1alpha (Ef1alpha; 1028 bp); (2) tektin (tektin; 715 bp); (3) wingless (wg; 405 bp); (4) ribosomal protein L5 (RpL5; 722 bp, exons 1, 2, 3, and introns 1 and 2); and (5) mitochondrial cytochrome oxidase I, II (Co1 and Co2 and intervening leucine tRNA; 1599 bp). The results show incongruence between some genetic loci, although when alternate topologies are compared statistically it was generally true that one topology was supported by a majority of loci sampled. This highlights the need to sample widely across the genome in order to obtain a well-supported phylogenetic hypothesis. A combined evidence topology is presented based on a Bayesian analysis of all the gene regions, except the fast-evolving RpL5. The resulting hypothesis is concordant with the most probable relationships determined from our topological comparisons, although in some parts of the tree relationships remain weakly supported. The tree suggests diversification has largely occurred within biogeographic regions such as Central America, the Amazon, the southern and northern Andes, with only occasional dispersal (or vicariance) between such regions. This phylogenetic hypothesis can now be used to investigate patterns of diversification across the genus, such as the potential role of color pattern changes in speciation.

Animals↗

Phylogeny of Tubificidae (Annelida, Clitellata) based on mitochondrial and nuclear sequence data.

The tubificid clitellates are a common component in the freshwater bottom fauna and are also the most abundant oligochaete group in marine habitats. There are over 800 described species classified in six subfamilies; Tubificinae, Limnodriloidinae, Rhyacodrilinae, Telmatodrilinae, Phallodrilinae, and Naidinae. In this study we examine the phylogenetic relationships in Tubificidae using a combination of mitochondrial 16S rDNA and nuclear 18S rDNA sequence data. Sequences were obtained from five outgroup and 56 ingroup taxa, including five of the six subfamilies of Tubificidae. The data were analysed by maximum parsimony and Bayesian inference. The resulting tree topologies are virtually without conflict. Several associations traditionally recognized within the family Tubificidae are supported, in the Bayesian analysis including a sister group relationship between Tubificinae and Limnodriloidinae. The results also indicate that Rhyacodrilinae is polyphyletic--some of its members (Heterodrilus spp.) fall into a clade with Phallodrilinae, all other groups with Naidinae. Naidinae is also polyphyletic with two rhyacodriline genera, Monopylephorus and Ainudrilus, nested within. Most of the tubificid genera included in the study are supported as monophyletic; however, Tubifex and Limnodriloides are refuted, and Tubificoides is unresolved from other tubificine taxa.

Animals↗